Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales

Definitions

• the present invention relates to a process for producing amino acids such as L-threonine and L-lysine by fermentation.
• the amino acids are widely utilized in the fields of drugs, foodstuff, animal feed, etc.
• amino acids such as L-threonine and L-lysine can be produced in high yields and at low cost by culturing, in a medium containing methanol as a major carbon source, a microorganism which belongs to the genus Methylobacillus and which has an ability to produce the amino acid(s) and resistance to at least one member selected from the group consisting of L-threonine, L-lysine and an amino acid analogue, said microorganism being obtained by mutation of a parent strain which belongs to the genus Methylobacillus and which has an enhanced sensitivity to at least one of an antibiotic and an amino acid analogue; allowing the amino acid(s) to accumulate in the culture; and recovering the amino acid(s) therefrom.
• a microorganism which belongs to the genus Methylobacillus and which has an ability to produce the amino acid(s) and resistance to at least one member selected from the group consisting of L-threonine, L-lysine and an
• any microorganism can be used in the present invention, so long as it has the following properties: (1) it belongs to the genus Methylobacillus; (2) it is obtained by mutation of a parent strain belonging to the genus Methylobacillus which has an enhanced sensitivity to at least one of an antibiotic and an amino acid analogue; (3) it has resistance to at least one member selected from the group consisting of L-threonine (hereinafter abbreviated as L-Thr), L-lysine (hereinafter abbreviated as L-Lys) and an amino acid analogue; (4) it can grow in a medium containing methanol as a major carbon source; and (5) it has the ability to produce amino acids, especially L-Thr or L-Lys.
• a mutant having such properties can be obtained by subjecting the parent strain to a conventional mutational treatment such as ultraviolet irradiation, a treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), or the like.
• antibiotics examples include kanamycin, ampicillin and streptomycin.
• amino acid analogue examples include ⁇ -amino- ⁇ -hydroxyvaleric acid (hereinafter abbreviated as AHV), S-2-aminoethyl-L-cysteine (hereinafter abbreviated as AEC) and DL-4,5-transdehydrolysine (hereinafter abbreviatedas DHL).
• AHV ⁇ -amino- ⁇ -hydroxyvaleric acid
• AEC S-2-aminoethyl-L-cysteine
• DHL DL-4,5-transdehydrolysine
• Wild strains of bacteria belonging to the genus Methylobacillus have a low sensitivity to an amino acid analogue, so that it is difficult to confer resistance to the amino acid analogue on the wild strains and isolate mutants which are released from metabolic regulation.
• the reason why these strains have a low sensitivity to the amino acid analogue is believed to be their poor membrane permeability to various chemicals. Therefore, amino acid leaky mutants are induced from the wild strains, and a strain which shows an enhanced sensitivity to various chemicals and an improved permeability to the chemicals is selected as a parent strain from the thus obtained mutants.
• the parent strain there may be used a strain belonging to the genus Methylobacillus and having an enhanced sensitivity to at least one member selected from antibiotics such as kanamycin, ampicillin and streptomycin and amino acid analogues such as AHV and AEC.
• antibiotics such as kanamycin, ampicillin and streptomycin and amino acid analogues such as AHV and AEC.
• mutants of wild strains such as Achromobater methanolophila ATCC 21452, Pseudomonas insueta ATCC 21276, Protaminobacter thiaminophagus ATCC 21371 and Methanomonas methylovora ATCC 21369 which have an enhanced sensitivity to the chemicals mentioned above.
• ATCC 21452 strain, ATCC 21276 strain, ATCC 21371 strain, ATCC 21369 strain, ATCC 21966 strain, ATCC 21967 strain and ATCC 21969 strain are hereinafter referred to as Methylobacillus sp. 1001, Methylobacillus sp. 1011, Methylobacillus. sp. 1006, Methylobacillus sp. 1003, Methylobacillus sp. K-015, Methylobacillus sp. K-038 and Methylobacillus sp. K-224, respectively.
• Methylobacillus sp. 1001 Methylobacillus sp. 1011, Methylobacillus sp. 1006 and Methylobacillus sp. 1003 to the chemicals
• these wild strains are subjected to a conventional mutational treatment such as NTG treatment.
• a specific example of the obtained mutant is Methylobacillus sp. iAlll. The procedure for obtaining the iAlll strain is shown below.
• Methylobacillus sp. 1011 is cultured in Ml medium having the following composition at 30° C. for 24 hours.
• the cultured cells are subjected to NTG treatment (500 mg/l, 30° C., 30 minutes) in a conventional manner and then smeared on Ml agar plate medium (Ml medium +1.5% agar) containing 0.1% Casamino acid (manufactured by Difco Laboratories) and 20 mg/l L-tryptophan. After cultivation at 30° C. for 3 to 14 days, colonies formed are picked up and isolated. The thus obtained mutants are inoculated into 3 ml of a seed medium in a test tube, followed by cultivation at 30° C. for 18 hours.
• compositions of the culturing media are shown below.
• Seed Medium (a) Composition of Seed Medium
• Fermentation Medium (b) Composition of Fermentation Medium
• the pH is adjusted with sodium hydroxide or hydrochloric acid.
• components other than methanol are dissolved and the solution is sterilized with steam at 120° C. for 15 minutes.
• methanol which has been passed through a membrane filter (manufactured by Millipore Co., 0.45 ⁇ m) for sterilization is added in the amount indicated.
• the cells and calcium carbonate are separated from the culture by centrifugation.
• the amino acids contained in the supernatant of the culture are analyzed with an amino acid analyzer (manufactured by Nippon Bunko Co., Ltd., high performance liquid chromatography, amino acid analysis system).
• the strain whose culture supernatant contains amino acids such as gutamic acid, aspartic acid, valine and alanine is selected as an amino acid leaky mutant.
• the thus obtained amino acid leaky mutant is examined for sensitivity to chemicals in the following manner.
• the amino acid leaky mutant is inoculated onto Ml agar plate media containing kanamycin sulfate [Km](manufactured by Meiji Seika Co., Ltd.), ampicillin [Ap](manufactured by Sigma Co., Ltd.), streptomycin sulfate [Sm](manufactured by Nakarai Pharmaceutical Co., Ltd.), AHV (manufactured by Sigma Co., Ltd.) and AEC (manufactured by Sigma Co., Ltd.) at various concentrations.
• Cultivation is carried out at 30° C. for 2 to 5 days to examine the growth and a strain having an enhanced chemical-sensitivity compared with the parent strain is selected.
• Methylobacillus sp. 1011 A strain having a higher chemical-sensitivity than that of the parent strain, Methylobacillus sp. 1011, is named Methylobacillus sp. iAlll.
• the minimum concentration (minimum inhibitory concentration) of each chemical at which the growth of representative strains is inhibited is shown in Table 2.
• the minimum inhibitory concentration was determined using Ml agar medium containing 50 mg/l phenylalanine.
• the microorganisms used in the present invention are mutants obtained by conferring the resistance to at least one member selected from L-Thr, L-Lys and the amino acid analogue on the strains belonging to the genus Methylobacillus described above.
• Such mutants can be obtained by subjecting the parent strains to a conventional mutation treatment such as a treatment with NTG, and then isolating strains which can grow in or on a medium containing at least one member selected from L-Thr, L-Lys and the amino acid analogue at the concentration at which the parent strains can not grow.
• Methylobacillus sp. iAlll is cultured in Ml medium containing 0.1% Casamino acid and 20 mg/l L-tryptophan at 30° C. for 24 hours.
• the obtained cells are subjected to NTG treatment (500 mg/l, 30° C., 30 minutes), in a conventional manner.
• the treated cells are smeared on Ml agar plate medium containing 5000 mg/l L-Thr and cultured at 30° C. for 3 to 14 days to obtain cooonies of L-Thr-resistant mutants growable thereon.
• the colonies are picked up and subjected to L-Thr and L-Lys production test.
• a mutant having a higher productivity of L-Thr and L-Lys than that of the parent strain is selected and named Methylobacillus sp. TA-47.
• DHL was synthesized by the process described in Journal of Biochemistry, 100, 21-25 (1986), with reference to the description in Journal of the American Chemical Society, 83, 2279-2281 (1961), and the product having a purity of more than 97% was used after purification.
• K-224 strain is used as the parent strain instead of iAlll strain and 1000 mg/l L-Thr is added to Ml agar medium containing 50 mg/l phenylalanine in place of 5000 mg/l L-Thr, whereby L-Thr-resistant mutants are obtained.
• a mutant having a higher L-Thr productivity than that of the parent strain is selected and named Methylobacillus sp. TR-26.
• K-224 strain is used as the parent strain instead of iAlll strain and 1000 mg/l L-Thr and 1000 mg/l AEC are added to Ml agar medium containing 50 mg/l phenylalanine in place of 5000 mg/l L-Thr, whereby mutants resistant to L-Thr and AEC are obtained.
• a mutant having a higher productivity of L-Thr and L-Lys than that of the parent strain is selected and named Methylobacillus sp. ATR-89.
• the obtained mutants were deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, on Jun. 22, 1990 under the Budapest Treaty.
• the accession numbers of the respective strains are shown in Table 3.
• the obtained mutants and their parent strains were cultured on Ml agar medium containing the chemicals(s) described below at 30° C. for 48 hours, and the degree of growth was observed. The results are shown in Table 4.
• microorganisms used in the present invention are cultured by the process generally used for culturing a methanol-assimilating microorganism.
• any of synthetic media and natural media may be used so long as it contains carbon sources, nitrogen sources, inorganic materials, and if necessary, organic trace components.
• methanol is mainly used and added to the medium at a concentration of 0.05 to 30%.
• Organic acids such as pyruvic acid and 2-ketoglutaric acid and natural organic components such as yeast extract, peptone and corn steep liquor, may be added to the medium at a concentration of 0.01 to 4%, if the growth of the microorganism used and/or the production of L-Thr and L-Lys can be promoted by the addition
• ammonium sulfate, ammonium chloride, ammonium acetate, ammonium nitrate, ammonium phosphate, ammonia gas, aqueous ammonia, urea, etc. may be added to the medium at a concentration of 0.1 to 8%.
• small quantities of the trace components such as potassium phosphate, sodium phosphate, magnesium sulfate, ferrous sulfate and manganese sulfate are generally added.
• the cultivation is carried out under aerobic conditions, for example, by shaking culture or submerged culture with aeration and agitation at a temperature of 24 to 37° C. and at pH 5 to 9, and is completed generally in 24 to 120 hours.
• the amino acids such as L-Thr and L-Lys can be recovered from the culture by removing the precipitates such as cells from the culture and subjecting the resulting supernatant to conventional means such as ion exchange, concentration and salting out.
• the cell-free culture supernatant is adjusted to pH 2 with hydrochloric acid and then passed through a strongly acidic cation exchange resin (manufactured by Mitsubishi Kasei Co., Ltd.).
• the adsorbed component is eluted with diluted aqueous ammonia and then ammonia is removed. After concentration, alcohol is added to the concentrate and the crystals formed during storage under cooling are collected to give L-Thr.
• the cell-free of the culture supernatant culture is adjusted to pH 7.0 with aqueous solution of sodium hydroxide and then passed through a cation exchange resin (manufactured by Mitsubishi Kasei Co., Ltd.).
• the adsorbed component is eluted with diluted hydrochloric acid and the fractions corresponding to L-Lys are collected.
• Alcohol is added to the combined fractions and the crystals formed during storage under cooling are collected to give LLys.
• Methylobacillus sp. TA-47 was inoculated into 3 ml of seed medium (a) in a test tube and cultured with shaking at 30° C. for 18 hours. Then, 1 ml of the culture obtained was added to 10 ml of fermentation medium (b) containing 2% methanol in a 50-ml large test tube and cultured with shaking at 30° C. Twenty four hours after the start of the cultivation, 2% methanol was further added and the cultivation was continued for further 24 hours.
• the cells and calcium carbonate were removed by centrifugation and the concentration of L-Thr contained in the resulting supernatant was determined with an amino acid analyzer (manufactured by Nippon Bunko Co., Ltd., high performance liquid chromatography, amino acid analysis system).
• TA-47 strain was cultured in the same manner as in Example 1.
• the resulting culture supernatant (800 ml) was adjusted to pH 2 with hydrochloric acid and then passed through a column packed with strong cation exchange resin, DIAION SKlB (H type)(manufactured by Mitsubishi Kasei Co., Ltd.). After the column was washed with water, the component adsorbed onto the resin was eluted with 2 N aqueous ammonia The fractions containing L-Thr were combined and concentrated under reduced pressure Ethanol was added to the concentrate and the mixture was cooled to 4° C. to form crystals The crystals were collected and dried to give 1.25 g of L-Thr crystals having a purity of 98% or more.
• Methylobacillus sp. strains 1011, iAlll, TA-47, DA-35, AL-76, 1006, K-224 and ATR-89 were respectively cultured in the same manner as in Example 1.
• the concentration of L-Lys contained in the resulting culture supernatant was determined with the amino acid analyzer.
• Two loopfuls of Methylobacillus sp. AL-76 was inoculated into 25 ml of seed medium (a) in a 300-ml Erlenmeyer flask and cultured with shaking at 30° C. for 18 hours. The whole seed culture was transferred to a 2-l Erlenmeyer flask containing 225 ml of seed medium (a). Cultivation was carried out with shaking at 30° C. for further 18 hours.
• the whole of the resulting seed culture (250 ml) was inoculated into 2.25 l of fermentation medium (b) in a 5-l fermentor (manufactured by Mitsuwa Biosystem Co., Ltd.). Cultivation was carried out at 30° C. with agitation (600 rpm) and aeration (2.5 l/min). During the cultivation, pH of the medium was automatically adjusted to 6.8 with 6 N NH 4 OH solution. Methanol was added at a concentration of 0.5% at the start of the cultivation and then continuously added in such an amount that the concentration of 0.5% is maintained, using a perista pump (manufactured by Ato Co., Ltd.).
• AL-76 Strain was cultured in the same manner as in Example 4.
• the culture supernatant (1200 ml) obtained by centrifugation was adjusted to pH 7.0 with sodium hydroxide and then passed through a column packed with strong cation exchange resin, DIAION SKlB (NH 3 type)(manufactured by Mitsubishi Kasei Co., Ltd.). After the column was washed with water, the component adsorbed onto the resin was eluted with 1 N hydrochloric acid. The fractions containing L-Lys were combined and concentrated under reduced pressure. Ethanol was added to the concentrate and the mixture was cooled to 4° C. to form crystals The crystals were collected and dried to give 1.88 g of crystals of L-Lys hydrochloride having a purity of 96% or more.

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• 1990
  • 1990-08-02 JP JP2205567A patent/JP3046332B2/ja not_active Expired - Fee Related
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